Peptides from the E7 protein of human papilloma viruses 16 and 18 for detecting and/or diagnosing cervical and other human papilloma virus associated cancers

ABSTRACT

An isolated protein sequence or peptide from the E2, E6 or E7 early coding region of human papillomavirus (HPV) that is soluble in an aqueous medium, and characterized by a relative paucity of tryptophan, methionine and cysteine residues, and a relative abundance of glycine and asparagine residues. Also disclosed are isolated protein sequences or peptides from the E2, E6 or E7 early coding regions of HPV 16 and 18 and methodologies for detecting or diagnosing cancer or cellular abnormalities. Detection or diagnosis of Cancer or cellular abnormalities may include detecting or diagnosing pre-cancerous or pre-malignant conditions, cervical dysplasia, cervical carcinoma, koilocytosis, hyperkeratosis, intraepithelial lesions, and other cancers. A methodology for detecting or diagnosing cancer or cellular abnormalities comprises the steps of (1) reacting a sample of body fluid or tissue with isolated protein sequences or peptides; (2) forming an antibody-peptide complex; and (3) detecting the antibody-peptide complex.

RELATED APPLICATIONS

This application claims the benefit of Divisional U.S. patentapplication Ser. No. 10/612,818, filed Jul. 1, 2003, now U.S. Pat. No.6,933,123 and entitled “PEPTIDES FROM THE E2, E6, AND E7PROTEINS OFHUMAN PAPILLOMAVIRUSES 16 AND 18 FOR DETECTING AND/OR DIAGNOSINGCERVICAL AND OTHER HUMAN PAPILLOMAVIRUS ASSOCIATED CANCERS” which claimsthe benefit of U.S. Provisional Application Ser. No. 60/394,172, filedJul. 2, 2002, and entitled “NOVEL PEPTIDES FROM THE E2, E6 AND E7PROTEINS OF HUMAN PAPILLOMAVIRUSES 16 AND 18 FOR DIAGNOSING OR DETECTINGCERVICAL AND OTHER HUMAN PAPILLOMAVIRUS ASSOCIATED CANCERS,” and CIP ofco-pending U.S. patent application Ser. No. 09/828,645, filed Apr. 5,2001, now U.S. Pat. No. 6,743,593 and entitled “IMMUNOLOGICALMETHODOLOGY FOR DISCERNING HUMAN PAPILLOMAVIRUS,” which are incorporatedherein by reference.

BACKGROUND

1. Field of the Invention

The present invention relates generally to the field of peptidesreactive with antibodies formed against human papillomavirus (HPV). Somehave termed this type of peptide as antigenic or immunoreactive. Moreparticularly, the invention relates to peptides isolated, purified orderived from the early coding region of the E2, E6, and E7 oncoproteinsof HPV and method for use for the detection and/or diagnosis of HPVassociated epithelial cell abnormalities, precancerous conditions andcancers via an immunoassay.

2. The Background Art

The human papillomaviruses (HPV), named because certain types inducewarts or papillomas, cause virtually all cervical cancers (Nobbenhuis etal., “Relation of human papillomavirus status to cervical lesions andconsequences for cervical-cancer screening: a prospective study,” TheLancet, 354:20-25, 1999; Cuzick et al., “A systematic review of the roleof human papilloma virus (HPV) testing within a cervical screeningprogramme: summary and conclusions,” British Journal of Cancer,83:561-565,2000). These encompass not only squamous cell carcinomas(Nobbenhuis et al., 1999) but also adenocarcinomas (Pirog et al.,“Prevalence of human papillomavirus DNA in different histologicalsubtypes of cervical adenocarcinoma,” American Journal of Pathology,157:1055-1062, 2000). These viruses are also strongly associated withvulvar and vaginal carcinomas (Frisch et al., “Humanpapillomavirus-associated carcinomas in Hawaii and the mainland US,”Cancer 88:1464-1469, 2000; Sugase et al., “Distinct manifestations ofhuman papillomaviruses in the vagina,” International Journal of Cancer,72:412-415, 1997), as well as cancers of the anus (Frisch et al., 2000)and penis (Gregoire et al., “Preferential association of humanpapillomavirus with high-grade histologic variants of penile-invasivesquamous cell carcinoma,” Journal of the National Cancer Institute,87:1705-1709, 1995).

Moreover, HPV may be responsible for certain carcinomas in the head andneck region (Mellin et al., “Human papillomavirus (HPV) DNA in tonsillarcancer: clinical correlates, risk of relapse, and survival,”International Journal of Cancer, 89:300-304, 2000; Zumbach et al.,“Antibodies against oncoproteins E6 and E7 of human papillomavirus types16 and 18 in patients with head-and-neck squamous-cell carcinoma,”International Journal of Cancer, 85:815-818, 2000), seem associated withthe more deadly melanomas (Dreau et al., “Human papillomavirus inmelanoma biopsy specimens and its relations to melanoma progression,”Annals of Surgery, 231:664-671, 2000), and could play a role in lungcarcinomas (Soini et al., “Presence of human papillomavirus DNA andabnormal p53 protein accumulation in lung carcinoma,” Thorax 51:887-893,1996) and perhaps other cancers.

Cervical cancer is the second most common cancer among women worldwide.Each year about 450,000 women worldwide are diagnosed with cervicalcancer, and nearly 300,000 women die of this disease. Since the adventof organized cervical cancer screening by cytology fifty (50) years ago,the mortality rate of cervical cancer has dramatically decreased indeveloped countries. In fact, cervical cancer may be consideredpreventable. In this regard, an important key to prevention is thetimely identification and management of precancerous lesions andotherwise early cancers through accessible and affordable screeningprograms and methodologies.

At present, about twelve percent (12%) of female cancers worldwide aredue to HPV infections of the cervix. There is consensus among themedical community that oncogenic HPV detection would be an effective wayto identify cancer victims or those at high risk for the disease.Notably, HPV detection would facilitate earlier detection of cancer orcellular abnormalities suggesting cancer at a point in time when thecancer or cellular abnormalities exists at a more readily curable stage.

A primary methodology for public health screening for cervical cancerhas been the Papanicolaou (Pap) smear. For a variety of reasons, thePapanicolaou smear is less than an ideal screening test. Drawbacks mayinclude difficulty of obtaining samples, high rate of false negatives(up to twenty percent (20%)), and requirements for specialized labsstaffed by highly trained personnel. Nucleic acid screening methods havebeen developed by those skilled in the art, but are not ideal primarilydue to their high cost and like requirement for highly trainedpersonnel. Another assay developed by those skilled in the art involvesthe so-called “DNA Hybrid Capture.” This method, however, tends tosuffer from high cost and sampling difficulties, thus making it somewhatdisadvantageous as an ideal screening test.

Recently, those skilled in the art have developed methodologies forexamining the utility of HPV for diagnostic purposes. More particularly,IgA, IgG and IgM antibodies raised against HPV have been used in thedetection of infection with HPV and for diagnosing carcinoma orpre-stages thereof. Using these prior art techniques, immunoreactivepeptides isolated from HPV have been defined to have an epitope which isreactive with human sera. The prior art does not, however, disclose thepeptides of the present invention, nor teach that multiple combinationsof antibody-epitope complexes could be contemplated to producediagnostic assays with improved sensitivity and/or specificity.

Such prior art methods for cancer screening, are limited by their cost,sampling procedures, accuracy, equipment and personnel requirements.Therefore, and as readily appreciated by those skilled in the art, lowcost, simple, sensitive and specific assays that can be performed onreadily obtainable bodily samples would be a significant advancement inthe art. Such assays are disclosed and claimed herein.

BRIEF SUMMARY AND OBJECTS OF THE INVENTION

A primary object of the present invention is to provide novel antibodyreactive protein sequences or peptides isolated, purified or derivedfrom the HPV 16 and 18 E2 protein and the HPV 16 and 18 E6 and E7oncoproteins.

It is a further object to provide these peptides in a chemically pureform.

A further object to provide a simple, rapid, less expensive and moresensitive test for detecting or diagnosing not only HPV infections, butalso most, if not all, HPV associated neoplasms.

It is a still further object is to provide antigens for use in HPVinoculums that will induce antibody production and killer T cellactivity.

It is also an object of the present invention to provide novel antibodyreactive protein sequences or peptides isolated, purified or derivedfrom HPV genotypes 16, 18, 31, 33, 35, 45, 51, 52, 56 and 58.

In addition, it is an object of the present invention to provide novelantibody reactive protein sequences or peptides isolated, purified orderived from HPV further comprising one or more additional glycineresidues added at a carboxyl terminal residue end of the proteinsequence or peptide.

It is a still further object of the present invention to provide novelantibody reactive protein sequences or peptides isolated, purified orderived from HPV further comprising one or more additional asparagineresidues added at a carboxyl terminal residue end of the proteinsequence or peptide.

It is another object of the present invention to provide novel antibodyreactive protein sequences or peptides isolated, purified or derivedfrom HPV further comprising a combination of glycine and asparagineresidues added at a carboxyl terminal residue end of the proteinsequence or peptide.

Further, it is an object of the present invention to provide a methodfor detecting or diagnosing cancer or cellular abnormalities comprisingthe steps of reacting a sample of body fluid or tissue likely to containantibodies with one or more protein sequences or peptides isolated fromthe E2, E6 and E7 early coding regions of HPV; forming anantibody-peptide complex comprising an antibody and a protein sequenceor peptide isolated from the E2, E6 and E7 early coding region of HPV;and detecting said antibody-peptide complex.

It is a still further object of the present invention to provide novelantibody reactive protein sequences or peptides isolated, purified orderived from HPV further comprising substitution of one or more cysteineresidues with one or more carboxymethylcellulose residues.

Additionally, it is an object of the present invention to provide amethod for detecting or diagnosing cancer or cellular abnormalitiesthrough detection or diagnosis of an HPV Epitope.

It is also an object of the present invention to provide a method fordetecting or diagnosing cancer or cellular abnormalities throughdetection or diagnosis of HPV associated cell abnormalities.

It is a further object of the present invention to provide a method fordetecting or diagnosing cancer or cellular abnormalities throughdetection or diagnosis of HPV associated precancerous or premalignantconditions.

It is a still further object of the present invention to provide amethod for detecting or diagnosing cancer or cellular abnormalitiesthrough detection or diagnosis of cervical dysplasia.

Additionally, it is an object of the present invention to provide amethod for detecting or diagnosing cancer or cellular abnormalitiesthrough detection or diagnosis of cervical carcinoma.

It is also an object of the present invention to provide a method fordetecting or diagnosing cancer or cellular abnormalities throughdetection or diagnosis of adenocarcinoma of the uterine cervix.

Further, it is an object of the present invention to provide a methodfor detecting or diagnosing cancer or cellular abnormalities throughdetection or diagnosis of HPV associated precancerous or premalignantconditions.

It is a still further object of the present invention to provide amethod for detecting or diagnosing cancer or cellular abnormalitiesthrough detection or diagnosis of cervical dysplasia.

Consistent with the foregoing objects, and in accordance with theinvention as embodied and broadly described herein, one presentlypreferred embodiment of the present invention comprises novel peptides,the sequences of which were isolated from careful analysis of the earlycoding regions of the E2, E6 and E7 oncoproteins of HPV 16 and 18. Thepeptides may lend themselves to a highly sensitive and specificdiagnostic immunoassay. Antibodies to the E2, E6 and E7 oncoproteins maybe found in those individuals infected with HPV associated neoplasms.

The peptides of the present invention, ranging in size, prior to anymodifications, from about 17 amino acid residues to about 23 amino acidresidues may be readily synthesized by chemical means and may beobtained at purities exceeding 95%. Although the peptides may beobtained by other means, in their pure form there is typically a muchreduced likelihood for undesirable cross reactivity with randomantibodies. Hence, the pure peptides of the present invention may lendthemselves to diagnostic immunoassays of high specificity. One presentlypreferred embodiment of a diagnostic immunoassay method of the presentinvention may include the steps of: (1) taking a sample of body fluid ortissue likely to contain antibodies; (2) if antibodies are present,reacting the sample with one or more of the peptides of the presentinvention; and (3) assaying the reacted sample for the presence of anantibody-peptide reaction.

Immunoassays employing the peptides isolated, purified or derived fromthe E6 and the E7 oncoproteins of HPV 16 and 18 may serve as reliableindicators that HPV associated malignancy or premalignant celltransformation has taken place. Likewise, immunoassays employing certainpeptides isolated, purified or derived from the E2 region of HPV 16 and18 may also be reliable indicators of HPV associated infection,malignacy or prelmalignant cell transformation.

One of the most useful advancements or aspects of the present inventionis in diagnosing cervical carcinoma, both squamous cell andadenocarcinoma, as well as any epithelial cell abnormality associatedwith oncogenic HPV infection including, but not limited to,koilocytosis; hyperkeratosis; precancerous conditions encompasssingintraepithelial neoplasias or intraepithelial lesion; high-gradedysplasias; and invasive or malignant cancers. Besides its utility incervical cancer diagnoses, finding antibodies to peptides isolated,purified or derived from the HPV E2, E6 and E7 oncoproteins of HPV 16and 18 may be valuable for detecting head and neck cancers, small celllung cancers, penile and anal carcinomas, melanoma and otherprecancerous or cancerous states caused by or otherwise associated withHPV.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects and features of the present inventionwill become more fully apparent from the following description andappended claims, taken in conjunction with the accompanying drawings.Understanding that these drawings depict only typical embodiments of theinvention and are, therefore, not to be considered limiting of itsscope, the invention will be described with additional specificity anddetail through use of the accompanying drawings in which:

FIG. 1 represents a table showing the results of Pap smear cytology anddiagnostic immunoassay applying HPV DNA Hybrid Capture on subjects withand without HPV associated dysplasia or otherwise premalignantconditions as well as cancer;

FIG. 2 represents a table further showing the results of a diagnosticimmunoassay applying the peptides of the present invention on subjectswith and without HPV associated dysplasia or otherwise premalignantconditions as well as cancer.

DESCRIPTIONS OF THE PREFERRED EMBODIMENTS

It will be readily understood that the isolated protein sequences andmethodologies of the present invention, as generally described andillustrated in the Figures herein, could be arranged and designed in awide variety of different configurations. Those of ordinary skill in theart will, of course, appreciate that various modifications to thedetails herein may be made without departing from the essentialcharacteristics of the invention, as described. Thus, the following moredetailed description of the embodiments of the isolated proteinsequences and methodologies of the present invention, as represented inFIGS. 1 and 2, is not intended to limit the scope of the invention, asclaimed, but it is merely representative of the presently preferredembodiments of the invention.

HPV exists as different genetic types or genotypes, designated bynumbers, concerning which only a subset is oncogenic or cancer causing.Over 100 HPV genotypes have been identified. These HPV genotypes aresometimes referred to as HPV strains or types and are often designatedor referred to by number only or by “HPV #”, where “#” is the number ofthe oncogenic or cancer causing genotype. Cancers overwhelmingly stemfrom HPV 16 and 18, but may also be associated with HPV 31, 33, 35, 45,51, 52, 56 and 58. The virus infects cervical and other cells thatgenerally support virus propagation, where it may cause abnormalcellular changes that can lead to life threatening malignancies.

HPV infection requires cells that are able to replicate their DNA,specifically those cells in the basal epidermal layer. HPV entry intothe basal epidermal layer occurs through microlesions that expose basalproliferating cells to the surface. The virus attaches to a cell surfacereceptor and gains entry into the cell cytosol. The infecting HPVparticle contains a closed-circular double-stranded DNA genome of 7000to 8000 base pairs composed of eight early transcribed open readingframes, E1 to E8, sometimes referred to as early coding regions andwhich are unequally represented among HPV genotypes, two late openreading frames and a noncoding long control region.

Much has been discovered about how HPV DNA integrates into hostchromosomes and how the E1 and E2 oncoproteins are involved with thisprocess. Relevance to immunological diagnostics is that antibodiesagainst E1 and E2 gene products may be evidence that HPV infection hasoccurred.

The manner by which HPV infection leads to cancer includes cellularinteractions with the E6 and E7 gene products. Stated another way, theE6 and E7 genes encode amino acids which are translated into proteinsequences or peptides, which then interact with cell structures orproteins and may lead to cancer. Gene products which may lead to tumoror cancer are often referred to as being oncogenic proteins oroncoproteins.

In host cells, E6 and E7 gene products form complexes with the cellularp53 and retinoblastoma tumor suppressing proteins regulating celldivision. By functionally neutralizing or inactivating these proteins,cells enter into the S phase of the cell cycle. The E7 oncoproteinfurther destabilizes cell control through its interaction with thecyclin-dependent kinase inhibitor protein, p21. These interactions setthe stage for controlling host cell proliferation and differentiation(i.e., transformation), a first step in the conversion of normal cellsto preneoplastic (i.e., pre-cancerous) cells and ultimately to the fullexpression of malignancy or cancer.

The E6 and E7 oncoproteins are constitutively expressed in tumor cells,and silencing these genes yields reversion of the malignant phenotype.Thus, the E6 and E7 gene products seem tumor-specific antigens, andpossible targets or probes for antibodies in immunological cancer testsas well as antigens in vaccines for controlling HPV-induced tumors.

Indeed, the E6 and E7 oncoproteins appear natural targets for antibodyproduction due to their consistent expression in cervical cancer cells.The response against the E7 oncoprotein in earlier studies had only beenmoderately disease specific, but E7 IgG and IgA have now been verifiedas strongly disease associated. Antibodies against the E6 and E7oncoproteins are at high levels in sera from cervical cancer patientscompared against non-cancer controls. Moreover, such antibodies seemdetectable by immunological means even when present in lesser amounts.Sensitivity for identifying HPV infections and possible cancersincreases with a combination of serological tests of multiple virusproteins. Hence, using both oncoproteins yields positive immunologicalresults with samples from cervical cancer patients.

In addition to the methodologies of the present invention, it may alsobe advantageous to employ an detection or diagnostic methodologyutilizing a combination of gene products, protein sequences or peptidesfrom two or more of the HPV E2, E6 and E7 early coding regions. Acombination of isolated protein sequences or peptides from HPV E2 withHPV E6 or E7 may allow for more sensitive and/or more specificmethodologies for detecting or diagnosing HPV virus integration(i.e.,infection) into a host cell as well as detecting or diagnosing HPVassociated cellular abnormalities. Alternatively, a combination ofisolated protein sequences or peptides from HPV E6 with HPV E7 may allowfor more sensitive and/or more specific methodologies for detecting ordiagnosing certain cellular abnormalities.

Antibodies against E2 may also be associated with premalignant statesand cancer (Stevenson et al., “Inverse relationship between theexpression of the human papillomavirus type 16 transcription factor E2and virus DNA copy number during the progression of cervicalintraepithelial neoplasias”, Journal of General Virology, 81:1825-1832,2001; Tonon et al., “Physical status of the E2 human papilloma virus 16viral gene in cervical preneoplastic and neoplastic lesions”, Journal ofClinical Virology, 21:129-134, 2001; Lindel et al., “Humanpapillomavirus positive squamous cell carcinoma of the oropharynx: aradiosensitive subgroup of head and neck carcinoma”, Cancer, 92:805-813,2001; Rosales et al., Antibodies against human papillomavirus (HPV) type16 and 18 E2, E6 and E7 proteins in sera: correlation with presence ofpapillomavirus DNA”, Journal of Medical Virology, 65:736-744, 2001;Sheets et al., “Immunotherapy of human cervical high-grade cervicalintraepithelial neoplasia with microparticle-delivered humanpapillomavirus 16 E7 plasmid DNA”, American Journal of Obstetrics andGynecology, 188:916-926, 2003), but specific details as to how the HPVE2 protein contributes to oncogenesis still remain obscure (Dong et al.,“Human papillomavirus type 11 E2 proteins repress the homologous E6promoter by interfering with the binding of host transcription factorsto adjacent elements”, Journal of Virology, 68:1115-1127, 1994).

The protein sequences or peptides of the present invention may beisolated, purified or derived from the early coding regions of the E2,E6, and E7 oncoproteins of HPV 16 and 18. The isolation of the peptidesof the present invention may be based on their ability to react withantibodies formed in a host infected with oncogenic HPV. Among thespecific factors used in the selection process is solubility in aqueoussolution or hydrophilic nature. It was assumed that hydrophilic regionsof the oncogene product protein were more likely oriented toward thesurface of the complete protein under natural or native conditions, andthat such consequently include antigenic regions against which antibodyreactivity would most likely occur.

Antigenic describes a substance that the body regards as foreign orpotentially dangerous and against which the body produces an antibody.Typically, an antigen is composed of a protein or peptide. An antibodyis a specialized protein produced in the lymphoid tissue in response tothe presence of a particular antigen. The antibody will attack theantigen and render it harmless. The antibody attack typically leads tothe formation of an antigen-antibody complex and is ofteninterchangeably used with the terms, antibody-peptide complex orpeptide-antibody complex and antibody-epitope complex.

Another factor is that an overall relative paucity of cysteine residuesin the amino acid sequence be maintained. More than one cysteine at theamino terminal residue was considered unacceptable. Attempts were alsomade to limit cysteine elsewhere along the peptide, since multiplecysteines could portend circumstances precluding usefulness in animmunoassay. Peptides with cysteine are more susceptible to oxidation ata neutral pH, and the propensity for cysteines to undergo sulphydrylformation could lead to a dimerization that impedes antibody binding.

In addition, there was looked for an overall paucity of tryptophan, dueto the elevated oxidative potential of this amino acid. By paucity, itis meant that there are as few occurrences as possible and by abundance,it is meant that there is no limit on the number of occurrences in thesequence. A still more preferred embodiment of the peptide compositionsincludes up to eight additional amino acid residues attached to thecarboxyl terminal residue where those residues are any combination ofglycine and asparagine. Additional glycine and asparagine amino acidresidues may help orient the peptide in aqueous medium such that bindingto antibody may be enhanced.

Eight specific protein sequences or peptides ranging from 17 to 23 aminoacid residues in length, may be isolated, purified or derived from theE2, E6 and E7 oncoproteins of HPV 16 and HPV 18, as defined hereinbelow.Sequence Identification Number (SEQ. ID. No.) 1 was derived from the E2Region of HPV 18. Decoded and using the standard three letter acronym,Sequence Number 1, comprised of the 22 residues making up amino acids219 through 240 of the HPV 18 E2 oncoprotein, is as follows, with thesequence beginning at the amino terminal residue and ending at thecarboxyl terminal residue:

(SEQ. ID. NO.1) Lys Gln Leu Gln His Thr Pro Ser Pro Tyr Ser Ser Thr ValSer Val 1                5                  10                  15 GlyThr Ala Lys Thr Tyr             20

Sequences numbers 2 and 3 are derived from the E2 early coding region ofHPV 16. Decoded using the standard three letter abbreviations, SequenceNumber 2, comprised of the 17 residues making up amino acids 112 through131 of the HPV 16 E2 oncoprotein, and Sequence Number 3, comprised ofthe 23 residues making up amino acids 187 through 209 of the HPV 16 E2oncoprotein, are as follows, with the sequence beginning at the aminoterminal residue end:

(SEQ. ID. NO. 2) Lys His Gly Tyr Thr Val Gln Phe Asp Gly Ile Cys Asn ThrMet His 1                 5                 10                  15 Tyr(SEQ. ID. NO. 3) His Ala Gly Gly Gln Val Ile Leu Cys Pro Thr Ser Val PheSer Ser Asn 1                 5                  10                  15Glu Val Ser Ser Pro Glu         20

Sequence number 4 is derived from the E6 early coding region of HPV 16.Decoded using the standard three letter abbreviations Sequence Number 4,comprised of the 22 residues making up amino acids 62 through 83 of theHPV 16 E6 oncoprotein, is as follows, with the sequence beginning at theamino terminal residue end:

(SEQ. ID. NO. 4) Arg Asp Gly Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu LysPhe Tyr 1                5                  10                  15 SerLys Ile Ser Glu Tyr             20

Sequence number 5 is derived from the E6 early coding region of HPV 18.Decoded using the standard three letter abbreviations, Sequence Number5, comprised of the 22 residues making up amino acids 67 through 88 ofthe HPV 18 E6 oncoprotein, is as follows, with the sequence beginning atthe amino terminal residue end:

(SEQ. ID. NO. 5) Lys Cys Ile Asp Phe Gly Ser Arg Ile Arg Glu Leu Arg HisTyr Ser 1                 5                  10                  15 AspSer Val Tyr Gly Asp             20

Sequences numbers 6 and 7 are derived from the E7 early coding region ofHPV 16. Decoded using the standard three letter abbreviations, SequenceNumber 6, comprised of the 21 residues making up amino acids 26 through46 of the HPV 16 E7 oncoprotein, and Sequence Number 7, comprised of the21 residues making up amino acids 67 through 88 of the HPV 16 E7oncoprotein, are as follows, with the sequences beginning at the aminoterminal residue end:

(SEQ. ID. NO. 6) Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile AspGly Pro 1                5                  10                  15 AlaGly Gln Ala Glu             20 (SEQ. ID. NO. 7) Glu Thr Thr Asp Leu TyrCys Tyr Glu Gln Leu Asn Asp Ser Ser Ser1                5                  10                  15 Glu Asp GluIle Asp             20

Sequence number 8 is derived from the E7 early coding region of HPV 18.Decoded using the standard three letter abbreviations, Sequence Number8, comprised of the 22 residues making up amino acids 14 through 35 ofthe HPV 18 E7 oncoprotein, is as follows, with the sequence beginning atthe amino terminal residue end:

(SEQ. ID. NO. 8) His Leu Gln Pro Gln Asn Glu Ile Pro Val Asp Leu Leu CysHis Glu 1                5                  10                  15 GlnLeu Ser Asp Ser Glu             20

The use of the peptides in a diagnostic method is based on the fact thatantibodies to the native epitopes of the E2, E6, and E7 oncoproteins ofHPV 16 and 18 are found in those suffering from a variety of HPVassociated cell abnormalities that exist in neoplasms, from precancerousstates to malignancy. More particularly, such HPV associated cellularabnormalities or dysplasias may include, but are not necessarily limitedto, koilocytosis; hyperkeratosis; precancerous conditions encompassingintraepithelial neoplasias or intraepithelial lesions; high-gradedysplasias; and invasive or malignant cancers. Dysplasia generallyrefers to the presence or development of abnormal, premalignant orprecancerous cells. The malignant neoplasms associated with HPV arediscussed above.

The diagnostic method comprises taking a sample of body fluid or tissuelikely to contain antibodies. This sample is preferably easy to obtainand may be serum or plasma derived from a venous blood sample or evenfrom a finger prick. However, cervical secretions, cervical tissue,tissue from other body parts, or other bodily fluids are known tocontain antibodies and may be used as a source of the sample. Once thepeptide antigen and sample antibody are permitted to react in a suitablemedium, an assay is then performed to determine to presence of anantibody-peptide reaction.

The following example will illustrate the practice of the presentinvention in further detail. It will be readily understood by thoseskilled in the art that the following methods, formulations, andcompositions of novel peptides from the early coding regions of E2, E6and E7 of HPV 16 and 18 of the present invention, as generally describedand illustrated in the Examples herein, are to be viewed as exemplary ofthe principles of the present invention, and not as restrictive to aparticular structure or process for implementing those principles. Thus,the following more detailed description of the presently preferredembodiments of the methods, formulations, and compositions of thepresent invention, as represented in Example I, is not intended to limitthe scope of the invention, as claimed, but is merely representative ofpresently preferred embodiments of the invention.

EXAMPLE I

1. Synthesis of the Amino Acid Sequences or Peptides

While the peptides of the invention may be obtained by a variety ofprior art methods, including, but not limited to recombinant technologyand other biotechnology sources, chemical synthesis is the preferredmethod as it facilitates the accumulation of a sizable quantity ofpeptide in a substantially pure form, 95 to 99% by weight in the presentcase. The synthesis of peptides may be accomplished on a 0.25 scaleusing (9-fluorenyl) methoxycarbonyl (FMOC)-protected L-amino acids, withsuper acid-labile 2-chlorotrityl resin (Novabiochem, Nottingham, UK) asa solid support. A Resin may be preloaded into a reaction vessel, washedwith dimethyl formamide and then drained completely. To this resin maybe added 10 ml of 20% piperidine in dimethyl formamide. The mixture maythen be shaken for 5 minutes and drained. Another 10 ml of 20%piperidine in dimethyl formamide may be added, and the mixture shakenfor 30 minutes. After draining, the resin may be washed with dimethylformamide four times, and then once with dichloromethane. The resinbeads may be considered appropriately prepared if these turned blueusing the standard ninhydrin test.

For each amino acid a coupling solution may be prepared, as follows: 1mmol Fmoc Amino Acid of choice; 2.1 mL 0.45 M2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium-hexafluoro-phosphate/hydrobenzotriazole[1 mmol]; 348 μL of N,N-diisopropylethylamine [2 mmol]. The mixture maybe shaken for a minimum of 30 minutes. The reaction vessel may bedrained and the resin washed four times with dimethylformamide, and afinal time with dichloromethane. A standard ninhydrin test may beperformed to ascertain coupling of the amino acid. For each amino acid,coupling solution may be added to the resin in the appropriate order,with the coupling reaction repeated until all amino acids may be inplace along the peptide.

The completed peptide may be cleaved from the resin by reaction for twohours with a solution of 5% H2O, 5% phenol, 3% thioanisole, 3%ethanedithiol, 3% triisopropylsilane, 81% trifluoroacetic acid. Aftercleavage, the resin mixture may be filtered into coldmethyl-tbutyl-ether. The precipitated peptide may then be washed twicewith cold methyl-tbutyl-ether and dried under gaseous nitrogen. Themolecular weight of the peptide may be checked by Matrix-Assisted laserDesorption Time-of Flight Mass Spectrometry, and the purity by HighPerformance Liquid Chromatography using a C18300A 5 μ column. Thesynthesized peptide sequences may be about at a 95 to 99% level ofpurity, but it is emphasized that lesser levels may be consideredpossibly appropriate for assay purposes.

2. Storage of the Amino Acid Sequences

The manufactured amino acid sequences may be suspended in PBS at aboutpH 7.0 to a concentration of about 1 mg/mL and may be stored in sealedvials at about −20° C.

3. Maleic Anhydride Binding of the Amino Acid Sequences to Titer Plates

REACTI-BIND™ Maleic Anhydride Activated Polystyrene Plates (Pierce,Rockford, Ill.) may be employed. Each amino acid sequence may be dilutedto 12.5 μg/mL with coating buffer (100 mM sodium bicarbonate buffer, pH9.4). To each titer well, 100 μL (1.25 μg) of the diluted sequencesolution may be added. The plate may then be incubated for one hour atroom temperature with shaking. The plate may be emptied and residualliquid tapped onto a clean paper towel. Each well may be washed with 100μL wash buffer (0.1% bovine serum albumin and 0.05% Tween-20 inphosphate buffered saline, pH 7.0). This may be repeated for a total ofthree times. Each time, the plate may be emptied and residual liquidtapped onto a clean paper towel. To each well, 200 μL of blockingsolution (3% bovine serum albumin and 0.05% Tween-20 in phosphatebuffered saline, pH 7.0) may be added.

Blocking solution may be left in each well for one minute. The titerplate may then be emptied by inversion. Filling with blocking solutionand emptying may be done three times. Finished titer plates may be driedat room temperature and stored at 4° C. for up to four months.

4. Sample Collection

All samples may be taken from female subjects by physicians or physicianassistants during visits for gynecological examinations. Cotton swabsmay be used to gather endocervical cells. Cells for the ThinPrep Papsmear (Cytyc Corporation, Stamford, Conn.) may be dispersed in ThinPreppreservative solution. Cells for the HPV DNA Hybrid Capture assay(Digene Corporation, Silver Spring, Md.) may be suspended in the samemedium. Both the ThinPrep Pap smear and the HPV DNA Hybrid Capture assayare further elucidated below.

Venous blood may be obtained by prescribed phlebotomy methods, with a21- or 22-gauge double-pointed needle into a “red top” tube. A total of7-9 mL blood may be taken from each subject. After allowing 15-20minutes at room temperature for clot formation, the blood may becentrifuged at 2,500 g for 15 minutes. Serum may be separated byaspiration from the clotted cells, using a disposable pipette, dispensedinto Eppendorf tubes as 0.25-ml aliquots, for storage at −80° C.

5. Immunoassay

For negative samples, serum may be obtained from virgin females, ages 14and 15, and from adult sexually active, monogamous women, more than 25years old, with histories of no Pap smear abnormalities and DNAverification of HPV absence. Positive samples consisted of serumobtained from females diagnosed with cervical cancer or precancerousdisease. Subject and control sera may be diluted 1:25 with wash buffer(0.1% bovine serum albumin and 0.5% Tween-20 in phosphate bufferedsaline, pH 7.0). To each well, 100 μL of diluted serum may be added, andthe assay plate incubated for one hour at room temperature with shaking.Each well may then be rinsed three times, each with 200 μL wash buffer.Each rinse may be for five minutes. The plate may be emptied each timeby tapping residual liquid onto a clean paper towel.

To each well may be added 100 μL horseradish peroxidaseconjugated-mouse-anti-human IgG diluted 1:12,000 with wash buffer. Theassay plate may then be incubated for 1 hour at room temperature. Usingmultiple pipettes, each well may be rinsed with 200 μL wash buffer fourtimes. Each rinse may be for five minutes. Before each rinse, the platemay be emptied and residual liquid tapped on a paper towel.

To each well may be added 100 μL 3,3′,5,5′ tetramethylbenzidine, asubstrate for horseradish peroxidase. This may be incubated at roomtemperature until a visually obvious green-blue color develops,typically within 10-15 minutes, and the reaction stopped by placing 150μL 1.5 M sulphuric acid (H₂SO₄) into each well.

6. Comparisonal Tests—ThinPrep Pap Test and HPV DNA Hybrid Capture

ThinPrep Pap Test—The ThinPrep Pap test Pap smears may be used forcytological verification of the presence or absence of cellularabnormalities indicative of cancer or precancer. Allowed by the US Foodand Drug Administration as a replacement for the conventional Pap smear,the ThinPrep Pap Test overcomes the limitations of the conventionalmethod. By improving the way the sample slide is prepared, the ThinPrepPap Test may actually improve the quality of the test. In clinicaltrials, the ThinPrep Pap Test improved the detection of low-grade andmore severe lesions by 65% in screening populations and by 6% inhigh-risk populations. Its use also reduced the number ofless-than-adequate specimens by more than 50%.

Rather than smearing the cervical sample onto a slide as is done withthe conventional Pap smear, the cervical swab may be rinsed in a vial ofpreserving solution. The specimen may be sent to a certified clinicallaboratory, where an instrument, the ThinPrep 2000 processor, may beused to disperse and filter the contents to reduce blood, mucus, andinflammation. A thin, even layer of the cervical cells may then bemechanically deposited onto a slide, the result being a uniformpreparation of well-preserved cells ready for precise microscopicexamination. Slides may be microscopically examined and interpreted by aboard certified gynecological cytologist.

Hybrid Capture II HPV DNA Testing—The Hybrid Capture II HPV DNA test(Digene Corporation, Silver Springs, Md.) may be employed as acomparison to ELISA tests employing peptides of the invention. It isapproved by the U.S. Food and Drug Administration to test for oncogenicHPV DNA, as reflexive follow-up of a Atypical Squamous Cells ofUndetermined Significance (ASCUS) or other abnormal Pap results. Indeed,the Hybrid Capture II HPV DNA test discerns virus DNA and not cervicaldisease, but all precancerous and cancerous states include HPV DNApresence. The hybrid capture involves a molecular hybridization thatuses non radioactive probes with amplification of the detection of thehybrid ones for chemoluminescence. The material for analysis may bedenatured and reacts with specific genic probe forming hybrid RNA/DNAthat are captured by antibodies that cover the walls of the tube. Tofollow the hybrids immobilized, these may be reacted with specificantibodies against RNA/DNA conjugated with alkaline phosphatase. Forminga stable substratum, the nucleic acid hybrids may be detected bychemoluminescence via spectometry.

The test may be run according to the manufacturer's protocol using themicrotiter plate based format and probes for “high carcinogenic risk” or“oncogenic” HPV types. Human papillomavirus determinations may bequantitative, with samples producing readings of 1 or more times thepositive control (1 pg/mL HPV DNA or 5000 HPV genome copies per test)considered to contain virus DNA.

7. Visualization/Interpretation of Completed ELISA Tests

Presence of an antibody-peptide complex may be signaled by visualizingphysical-chemical changes that occur upon formation of anantibody-peptide complex. Physical-chemical changes may occur inconnection with oxidation reactions and other chemical reactions.Physical-chemical reactions may be detecting using a spectrophotometeror the like.

The bottom of the titer plate may be cleaned with 70% ethanol, and thetiter plate loaded into the spectrophotometer. Absorbance may be read at450 nm, with 100 mL of TMB solution plus 100 mL of 2N HCl used as ablank control.

8. Results

The results comparing Pap smear cytology, Digene HPV DNA assays, andimmunoassay according to the invention are shown in FIGS. 1 and 2.Referring now to FIGS. 1 and 2, sixteen female subjects were tested.Samples from women with a low pre-test probability by virtue of sexualhistory and/or Pap smear history were negative in all samples for alltests actually performed, evidenced by absorbance values that averaged0.19 compared against the 0.42 average for subjects with varied statesof disease, with the singular exception discussed below. This indicatesa low rate of false positives and a high negative predictive value.

Samples from subjects with a low pre-test probability by virtue ofsexual history and/or Pap smear history mostly had lower absorbancevalues, as stated above. However, Subject 6 was exceptional in terms ofits higher values for HPV E2 antibody-antigen complexes, averaging 0.42with 0.44 for SEQ ID NO 1, 0.32 for SEQ ID NO 2 and 0.49 for SEQ ID NO3. Subject 6 otherwise yielded absorbance values that averaged 0.21.These data are not indicators of infection or dysplasia. With no HPV DNAdetected in Subject 6, it is more likely that these elevated HPV E2absorbance values were vestiges of a previous but now cleared HPVinfection.

Samples from women with a high pre-test probability by virtue of provenclinical/pathological history largely showed immunoassays performedaccording to the invention as positive, indicated by higher absorbancevalues. Average absorbance values were 0.42 for serum from diseasedSubjects 8-16, and 0.47 with consideration of only those peptidesderived from the E6 and E7 oncoproteins. Furthermore, these values aresignificantly higher on average than absorbance values from non-diseasedor healthy subjects (p<0.001). These results point to a high positivepredictive value. Since in some cases one or more assays yieldedsomewhat lower absorbance values, the merit to employing combinations ofpeptides is demonstrated. The low false positives and high truepositives indicates a test of high sensitivity and high specificity forprecancerous disease and cancers.

Of central importance is that Subjects 15 and 16 had pathology provenadenocarcinoma of the cervix, that Subjects 13 and 14 had pathologyproven squamous cell carcinoma of the cervix, and that serum from thesesubjects were characterized by higher absorbance values. It is likewiseimportant that Pap smear scrutiny for Subject 15 resulted in no findingsof cell abnormalities in the face of significant cervical disease. ThatPap smear cytology can miss adenocarcinomas underscores the usefulnessof the peptides of the present invention.

It is likewise notable that serum from Subject 7 showed lowerabsorbencies for virtually all peptides of the present invention whencompared against results for Subjects 8-16. Although Subject 6 came witha pathology proven diagnosis of cell abnormalities, immunoassays usingpeptides of the invention were more in accord with the negative DNAresults. These data were considered to indicate cell abnormalitieshaving nothing to do with oncogenic virus and a consequent smalllikelihood of future disease progression to a more serious dysplasia orcancer. These results point out the further utility of the presentinvention as described herein.

From the above discussion, it will be appreciated that the presentinvention provides novel antibody reactive protein sequences or peptidesisolated from HPV 16 and 18 E2, E6 and E7 early coding regions. Inpreferred design, the novel protein sequences or peptides provide amarker for a simple, rapid, less expensive, more sensitive and morespecific test for detecting or diagnosing not only HPV infections, butalso most, if not all HPV associated cellular abnormalities,pre-cancerous cells, cancers and neoplasms.

Unlike the prior art, the present invention provides methodologies fordetecting or diagnosing cancer or cellular abnormalities by obtaining abody tissue sample, reacting the sample with a novel protein sequencesor peptides isolated from HPV 16 and 18 E2, E6 and E7 early codingregions to form an antibody-peptide complex; and detecting theantibody-peptide complex.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrative,and not restrictive. The scope of the invention is, therefore, indicatedby the appended claims, rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

The invention claimed is:
 1. A method for detecting or diagnosing cancer or cellular abnormalities, said method comprising the steps of: reacting a sample of body fluid or tissue likely to contain antibodies with a protein sequence or peptide isolated from the E7 early coding region of human papillomavirus (HPV) 16 as set forth in SEQ. ID. NO.: 6; forming an antibody-peptide complex comprising the isolated protein sequence or peptide and said sample antibodies; and detecting said antibody-peptide complex, wherein detecting the antibody-peptide complex indicates cancer or cellular abnormality, wherein said cellular abnormalities are selected from the group consisting of dysplasias, koilocytosis, hyperkeratosis, precancerous conditions, intraepithelial neoplasias, intraepithelial lesions, high-grade dysplasias, invasive cancers, malignant cancers, premalignant cells, and precancerous cells.
 2. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said isolated protein sequence or peptide comprises one or more additional glycine residues added at a carboxyl terminal residue of said isolated protein sequence or peptide.
 3. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said isolated protein sequence or peptide comprises one or more additional asparagine residues added at a carboxyl terminal residue of said isolated protein sequence or peptide.
 4. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said isolated protein sequence or peptide comprises a combination of glycine and asparagine residues added at a carboxyl terminal residue of said isolated protein sequence of peptide.
 5. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said cysteine residues of said isolated protein sequence or peptide is substituted with a carboxymethlycysteine residue.
 6. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said diagnostic method is directed to detecting or diagnosing an HPV epitope.
 7. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said HPV epitope is an antigenic region against which antibody reactivity would occur.
 8. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said diagnostic meted is directed to detecting or diagnosing an HPV associated cell abnormality.
 9. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said diagnostic method is directed to detecting or diagnosing an HPV associated precancerous or premalignant condition.
 10. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said diagnostic method is directed to detecting or diagnosing an HPV associated cancer.
 11. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said diagnostic method is directed to detecting or diagnosing cervical dysplasia.
 12. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said diagnostic method is directed to detecting or diagnosing cervical carcinoma.
 13. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said diagnostic method is directed to detecting or diagnosing cervical cellular abnormalities selected from the group consisting of koilocytosis, hyperkeratosis, precancerous conditions encompassing intraepithelial lesions, high-grade dysplasias, invasive cancers and malignant cancers.
 14. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said diagnostic method is directed to detecting or diagnosing adenocarcinoma of the uterine cervix.
 15. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said protein sequence or peptide isolated from the E7 early coding region comprises detection or diagnosis of premalignant cell transformation, a precancerous condition or cancer.
 16. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said detection step further comprises the step of visually inspecting said antibody-peptide complex for a color change.
 17. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 1, wherein said detection step further comprises inspecting said antibody-peptide complex for physical-chemical changes.
 18. The method for detecting or diagnosing cancer or cellular abnormalities as defined in claim 17, wherein said inspection step further comprises inspecting said antibody-peptide complex using a spectrophotometer. 